1. A 500 µL HepG2 cell suspension was grown in 96-well plates at 5 x 104 cells/well density.
2. Incubate the cells at 37oC; 5% CO2 for 24 hours.
3. Add 1mM Oleic Acid as much as 500 µL in each well to trigger cell fibrosis and incubate the cells for 24 hours at 37oC; 5% CO2.
4. 500 µL of test and control solutions were put into the wells. The test solutions used were:
- Control group, which was not given any treatment (Cb)
- Silymarin group, which was given silymarin (Sigma-Aldrich, SCM152) with 250 mcg/ml solution (C1)
- UC-MSC group with a 1:1 ratio in DMEM growth medium (C2)
- UC-MSC group in silymarin carrier solution (C3).
5. Incubate the cells at 37oC; 5% CO2 with incubation periods of 24 hours and 48 hours.
6. Addition of 100µl 1X Cell Extraction Buffer PTR and remove cells using mini cell scrapper.
7. Cells were collected in a 1.5 ml microcentrifuge tube.
8. A total of 50µl cells were then plated in each well of white 96-well plates.
9. At the same time, Lyophilized NFkB p65 Control Lysate and Cleaved Caspase-3 (Asp175) lyophilized standards were prepared by stepwise dilution.
10. A 50µl standard was added to the first 2 columns of the 96-well plate.
11. Add 50µl Antibody Cocktail for NFkB and Caspase for each sample and standard.
12. Cover the 96-well plates with a seal and incubate for 1 hour on a plate shaker and at room temperature.
13. Wash each well with Wash Buffer 3 times.
14. Add 100µl TMB Development Solution and incubate for 30 minutes in the dark for 1 hour on a plate shaker.
15. We add 100µl Stop Solution to each well and shake the plate to level the solution with a plate shaker.
16. The absorbance was reading at 450nm wavelength.